The continuing goal of this competitive renewal application is to elucidate how aging causes well defined deficits in brain neurotransmitter release, focusing on the cholinergic system. As we explore this problem further at the biochemical level, new insights are provided about the regulation of the neurotransmitter-release process and its modulation in normal as well as senescent animals. The focus of the next four years shifts from that of calcium- fluxes and potency generally to that of the modulatory role of protein kinase C in these release-deficits. Our results and others suggest that this enzyme is a nodal point for the effects of aging and membrane peroxidation on transmitter release. Specifically, we propose to answer the following questions: 1. Does aging affect protein kinase C activity in different brain regions and preparations similarly? 2. How does aging interfere with the phorbol ester-induced release of acetylcholine (ACh) and the translocation of protein kinase C? 3. Does aging affect the potency of calcium ionophores with respect to protein kinase C translocation? 4. Does infusion of protein kinase C directly into synaptosomes affect protein phosphorylation and ACh release? 5. Does aging affect the protein kinase C-mediated phosphorylation of proteins in synaptosomes in situ? 6. Does phosphatidylserine restore age-related deficits in protein kinase C translocation to normal? 7. Does exogenous synaptosomal membrane peroxidation mimic the effects of aging on protein kinase C activities as it does ACh release and membrane fluidity? These studies combine a variety of enzyme assays and pharmacological approaches used in my laboratory, along with a viral coat infusion procedure adapted to infuse enzymes or other macromolecules into nerve terminals. We will continue to use diluted synaptosomes as the preparation of choice for reasons described in the previous grant.